Hardness and elastic modulus values were calculated, and the time-dependent behavior of the samples was studied. Submersion of PCL and the graft copolymer in α-MEM suggested the deterioration of the measured mechanical properties as a result of the samples’ degradation. However, even after three days of degradation, the graft copolymer presented sufficient mechanical strength and elastic properties, which resemble those reported for soft tissues. The in vitro biological evaluation of the material clearly demonstrated that the CS-g-PCL copolymer supports the growth of Wharton’s jelly mesenchymal stem cells and tissue formation with a simultaneous material degradation. Both the mechanical and biological data render the CS-g-PCL copolymer appropriate as a scaffold in a cell-laden construct for soft tissue engineering.Tissue regeneration necessitates the development of appropriate scaffolds that facilitate cell growth and tissue development by providing a suitable substrate for cell attachment, proliferation, and differentiation. The optimized scaffolds should be biocompatible, biodegradable, and exhibit proper mechanical behavior. In the present study, the nanomechanical behavior of a chitosan- Heart failure and myocardial infarction are the most frequent death causes in the world . SurgicaThe last several years there has been a tremendous attempt to blend CS and PCL, although these materials are considered immiscible ,26,27,28graft-poly(ε-caprolactone) (CS-g-PCL) copolymer was investigated, and its potential use in soft tissue regeneration was assessed. One of our aims was to elucidate the effect of submersion in the cell culture medium, alpha Modified Eagle’s Medium, on the mechanical and physicochemical properties of the copolymer. Moreover, we examined in vitro the viability and proliferation of mesenchymal stem cells (MSCs) derived from the Wharton’s jelly (WJ) of umbilical cords on CS-g-PCL copolymer discs up to seven days in culture, and the morphology of the cell-material construct after one and four weeks in culture. To the best of our knowledge, this is the first report on the mechanical properties of a CS-g-PCL graft copolymer investigated by nanoindentation in the hydrated and dry states. Ongoing work, on the differentiation of WJ-MSCs under induction with a cardiogenic medium comprising oxytocin, explores the potential of combining the differentiated cells with 3D scaffolds made from this material for use in myocardium tissue engineering applications.In the present study, the nanomechanical behavior of a chitosan-wM) = 110–150 kDa), Tin (II) 2-ethylhexanoate (Sn(Oct)2), glycolic acid, sodium dodecyl sulfate (SDS), N,N’ dicyclohexylcarbodimide (DCC), and N-hydroxysuccinimide (NHS) were obtained from Sigma . All other chemicals and solvents were of analytical grade and were used without further purification. PCL functionalized with one terminal carboxylic acid moiety (PCL-COOH) was synthesized by ring opening polymerization as described previously  (Ca (CaMw) =g-PCL were pressed into discs with a diameter of 13 mm and a thickness of 1.5 mm using a SPECAC mechanical compacting press . A pressure of 15 tons was applied for 3 min for the preparation of the samples.CS, PCL-COOH, and CS-−1. Proton nuclear magnetic resonance (1H NMR) spectra were measured on a Bruker AMX-500 spectrometer . Trimethylsilyl propanoic acid (TSP) was used as an internal standard when the solvent mixture CF3COOD:D2O was used. The molecular weight (wM) and molecular weight distribution (MWD) of poly(ε-caprolactone) were determined by gel permeation chromatography (GPC) . The instrument was equipped with two PL mixed-D and mixed-E columns and a Waters 415refractive index detector operating at 35 °C. Calibration was based on a series of six narrow wM linear poly (methyl methacrylate) standards ranging from 850 to 342,900 g mol−1 and THF was used as the eluent at a flow rate 1 mL min−1. The thermal stability of CS-g-PCL, PCL-COOH, and CS was investigated by thermogravimetric analysis (TGA) using a Perkin Elmer Pyris Diamond thermogravimetry/differential thermal analyzer (TG/DTA) Perkin Elmer, Llantrisant, UK) instrument. In a typical measurement, ~10 mg of the disc samples were placed in a platinum holder and were heated under constant nitrogen flow from room temperature up to 650 °C at a heating rate of 10 °C/min.Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra were recorded on a Nicolet 6700 optical spectrometer . For each spectrum, 128 scans were collected in the range of 400–4000 cmg-PCL were investigated using a PL-DSC differential scanning calorimeter . All measurements were carried out under a nitrogen flow, while controlled cooling was achieved using liquid nitrogen. The temperature was varied between −100 °C and 200 °C for CS and CS-g-PCL, and between −100 °C and 125 °C for PCL-COOH. In all of the measurements, one cooling and two heating cycles were performed. The first heating cycle was performed to erase the thermal history of the samples, whereas the melting temperature and the enthalpy of fusion (ΔHf) were determined from the second heating cycle for all samples. Furthermore, X-ray diffraction (XRD) measurements were employed to examine the crystallinity of PCL-COOH, CS, and CS-g-PCL. XRD patterns were obtained on a PANanalytical X´pert Pro MPD powder diffractometer   using CuKa radiation (λ = 1.5418°).The thermal transitions of CS, PCL-COOH, and CS-g-PCL disc samples were disinfected with 70% ethanol in water, and then weighed for the degradation study. Each sample was incubated separately in a sealed tube with 20 mL α-MEM cell culture medium (pH 7.4) at 37 °C for 3 weeks. The CS-g-PCL and PCL samples were removed from the culture medium every 7 days, rinsed with distilled water, and dried for 1 h in an oven at 110 °C and at 37 °C, respectively, to effectively remove the water. Next, the samples were weighted with an accuracy of 0.01 mg before being placed back into a sterile tube with fresh culture medium. The % total weight loss for each sample was calculated after three weeks incubation in the culture medium. The values represent averages of triplicate experiments ± standard deviation (STDV).The PCL-COOH and CS-g-PCL and PCL-COOH disc samples, before and after degradation, were sputter-coated with a 10-nm thick layer of gold  and were visualized under a field-emission scanning electron microscope  at an accelerating voltage of 15 kV. The samples subjected to a three-week degradation process were first dehydrated in increasing ethanol concentrations and dried in a critical point drier  before being observed by FESEM.CS-Indentation testing of biological tissues and biomaterials possesses many challenges. The nanoindentation theory, analysis, and instrumentation have been developed and validated for smooth, solid, elastic, and elastic-plastic materials; however, biological materials, and in particularly soft tissues, represent a class of porous, hydrated, and viscoelastic materials with irregular geometries ,36,37,38g-PCL disc samples, whereas a fluid conospherical diamond indenter (50 μm tip radius) was used in the case of the α-MEM-submersed disc samples. Experiments were performed in a clean area environment with ~45% humidity and 23 °C ambient temperature. Using the Oliver and Pharr method, hardness (H), elastic modulus (E), and reduced modulus (Er) values were determined for the as-prepared (a) CS, and (b) CS-g-PCL samples. Tests at various maximum loads from 50 to 1000 μN with different hold times  at the maximum applied load and with an identical 10 s loading and unloading time were performed in order to investigate the time-dependent behavior of the materials. CS-g-PCL samples were also immersed in α-MEM at 25 °C and their mechanical behavior was studied after certain time periods . However, after immersion in α-MEM, the CS-g-PCL sample exhibited lower resistance and stronger adhesion of the tip, and thus the nanoindentation tests were conducted following the displacement control mode at different displacement depths (from 100 nm to 1000 nm), with a 10 s hold time and an identical 10 s loading and unloading time. All H, E, and Er values presented and calculated in this study are an average (±STDV) of six tests conducted in two different disc samples for CS and CS-g-PCL. It was also noticed that the CS sample became swollen within the first 2 h of immersion in α-MEM and completely dissolved in the aqueous medium after 24 h, prohibiting measurement of its mechanical properties.The nanomechanical characterization was performed with a Hysitron TriboLab® test instrument  equipped with a two-dimensional force displacement transducer. The transducer can apply loads in the range of 1–30,000 μN with a resolution of 1 nN, while the maximum penetration depth recorded is 3000 nm (3 μm) with a resolution of 0.04 nm. A Berkovich diamond indenter (100 nm tip radius) was used for the investigation of mechanical behavior of the as-prepared (a) CS, and (b) CS-2 for two passages.Wharton’s jelly mesenchymal stem cells (WJ-MSCs) isolation, expansion, and immunophenotypical analysis were performed as described previously . Briefly4) in complete α-MEM were seeded on the samples and placed in the cell culture incubator at 37 °C. Viable cell proliferation was performed with the colorimetric resazurin-based PrestoBlue® assay  using a calibration curve . T. T1H NMRCS-g-PCL ,43. Finag-PCL .TGA of the disc samples was employed to study the thermal stability of CS, PCL-COOH, and CS-g-PCL . The TGAed steps . The fired below . Finallyg-PCL were also investigated by DSC. g-PCL copolymer. The PCL-COOH homopolymer shows a melting temperature (Tm) at 53.8 °C and a crystallization temperature (Tc) at 27 °C, while, the DSC curve of CS does not exhibit any thermal transition in the range −100 to 200 °C. After grafting the PCL-COOH chains onto the CS backbone, a melting and crystallization transition appears for the graft copolymer at Tm = 54.7 °C and Tc = 28.6 °C, respectively. The above transitions are assigned to the PCL chains in the graft copolymer and further verify the successful grafting of PCL-COOH onto the CS backbone. Moreover, the enthalpy of fusion (ΔHf) for the PCL segment in the homopolymer and the graft copolymer was calculated from the respective DSC curves to be 87.86 J/g and 35.75 J/g, respectively. These values correspond to a crystallinity (Xc) of 65.8% for the PCL homopolymer and 26.8% for the PCL in the CS-g-PCL copolymer , and suggest a substantial reduction of the PCL crystallinity in the graft copolymer attributed to the complex copolymer architecture [The thermal transitions of CS, PCL-COOH, and CS-itecture . Finallyg-PCL graft copolymer. The XRD pattern of PCL-COOH shows three strong and sharp peaks at 21.5°, 22.1°, and at 23.9°, which are characteristics for PCL and are attributed to the (110), (111), and (200) crystallographic planes of PCL, respectively [g-PCL shows the two characteristics peaks of PCL at 21.5° and 22.1°. The intensity of these peaks is significantly reduced compared to those of the PCL homopolymer, verifying the decrease in the crystallinity of PCL in the graft copolymer, in good agreement with the DSC results discussed above. Moreover, the CS peak at 10.5° has disappeared in the XRD pattern of the graft copolymer, while the peak at 20.0° has become much broader, suggesting a decrease of the crystallinity of CS in the graft copolymer due to the presence of the grafted PCL chains along the CS backbone, which hinder the interchain hydrogen bonding interactions and lead to changes in the CS microstructure [XRD measurements were performed to determine the influence of the PCL-grafted chains on the crystallinity of the CS backbone. ectively . The XRDectively ,50. Finag-PCL samples. Although the two materials exhibit a similar degradation profile for the first two weeks, the total weight loss for the CS-g-PCL discoid samples was 35% ± 4%, which is significantly higher compared to that found for PCL-COOH (19% ± 2%) . The overall higher degradation rate of the copolymer was attributed to the presence of the hydrophilic CS segments, which facilitate water diffusion within the material.g-PCL disc samples  at various maximum depths. Taking into account the geometry of the Berkovich tip, the smooth load-unload curves indicate that there is no significant porosity in the samples synthesized and tested in this study [g-PCL samples show lower slopes of dP/dh  in the unloading step and higher values of maximum nanoindentation depths compared with pure CS samples. The higher plastic deformation is observed for the CS-g-PCL sample, because, at the same applied load, the indenter penetrated the sample at a higher indentation depth followed by higher dissipation of energy compared to the CS sample. The dissipation energy, as a quantitative reflection of the viscous effect, is due to the internal friction or plastic deformation energy inside the copolymer. The lower stiffness for the CS-g-PCL sample is attributed to the lower degree of crystallinity of CS chains in the graft copolymer. A similar effect has been reported earlier by [2 and –OH) and the carbonyl groups of PCL, which also resulted in the suppression of the PCL crystallization [Nanoindentation tests were performed at various applied loads and holding times reaching different penetration depths as shown in is study . The loalization ,30,33,52g-PCL copolymer determined at various maximum loads from 5 μΝ to 1000 μΝ, for 5, 10, and 20 s holding time, respectively, following the load control mode. In all three cases, the H, E, and Er values are higher at low penetration depths, whereas as the indenter penetrated further into the sample, the values decreased and finally reached a constant minimum value. It should be noted that the H, E, and Er values for low indentation depths have higher error bars probably due to the surface roughness that affects the calculation of the real contact area. As discussed above in g-PCL copolymer exhibited lower E values due to the lower degree of crystallinity of the CS chains as discussed above. Overall, it was found that the graft copolymer presented lower nanomechanical properties compared to the CS and higher compared to PCL-COOH homopolymer samples , and presents elastic values (≤1 kPa) very close to myocardium tissue in wet state. samples . CS reve samples . Ιn Tablg-PCL and CS samples   at maximum applied loads to study the creep behavior and the effect of holding time on the unloading segment of the CS- samples . Figure ed above ,30,33,52ed above .g-PCL copolymer sample became swollen and fractured after 4 weeks of immersion in the aqueous medium due to the presence of the hydrophilic CS component. g-PCL copolymer sample after the 1st, 2nd, and 3rd day of submersion in α-MEM. In g-PCL samples, respectively, are presented as a function of submersion time. As seen in g-PCL decrease rapidly in the first few hours of submersion due to the water uptake by the sample. In general, the kinetics of the scission of the ester bonds of aliphatic polyesters controls their hydrolytic rate, and an accelerated degradation is found in amorphous regions where the water molecules can penetrate faster within the disordered network of polymer chains compared to the crystalline ones [2 adsorption/desorption experiments, on the samples suggested that none of the materials used in this work exhibited any significant porosity. The fast degradation rate of the CS-g-PCL disc is attributed to the swelling of the material due to the presence of the hydrophilic CS component and the lower crystallinity of the PCL chains in the graft copolymer. However, despite the softening and the high degradation rate of the CS-g-PCL sample, it was observed that the graft copolymer presented adequate mechanical integrity under the applied load at the fully hydrated state for a period of 3 days.Following the nanoindentation tests performed on the as-prepared samples, all samples were submersed in α-MEM serum at 25 °C under static conditions, for a period of 3 days, and their nanomechanical properties were evaluated at certain time periods. As already stated above, the hydrophilic nature of CS resulted in the dissolution of the sample within 24 h after submersion in the medium. The CS-ine ones ,61. It wine ones ,63. Two ine ones . On the ine ones . For theg-PCL discs is whether they support cell adhesion, viability, proliferation, and tissue growth in vitro. We therefore investigated the viability of WJ-MSCs on CS-g-PCL discs following the good attachment of the cells on the material, and the cell proliferation after 3 and 7 days of culture. In a previous study, we showed a strong initial adhesion of WJ-MSCs seeded on two-dimensional films of the CS-g-PCL copolymer, and depicting a well-spread, flattened cell morphology onto the substrate [g-PCL discs as well as on a tissue-culture-treated polystyrene (TCPS) control after 1, 3, and 7 days in culture   c. Follow control .g-PCL discs after one week, and the formation of a dense tissue after four weeks accompanied by the successive degradation of the substrate, set the first in vitro study that renders the proposed cell-material construct suitable for tissue engineering applications. Still, for a long-term goal in a cardiac tissue application, there are several biological and functional related issues to be investigated. It is reported that a scaffold for cardiac repair should provide mechanical support and physical and biological cues and be degradable and capable of integration with host tissue [Our results on the proliferation increase of cells cultured on CS-t tissue . Additiot tissue . Decellut tissue . Also, Et tissue . Polysact tissue .According to the principle of tissue engineering, the degradation rate of a scaffold should be slower than the targeted tissue formation rate, since the strength of the scaffold must be sufficient to support the regeneration process. In our case, the degradation rate of the copolymer discs with a weight loss of 11% after one week, 18% after two weeks, and 35% after three weeks, could be beneficial for the formation of a soft tissue. Additionally, regenerating tissues require interaction with ECM components not only for mechanical support but also to acquire and maintain appropriate phenotypic and functional characteristics. Therefore, our future work will focus on the differentiation of the WJ-MSCs towards cardiac cells on the copolymer scaffolds, and the investigation of their in vitro and in vivo functionality.g-PCL graft copolymer was synthesized and used for scaffold fabrication for soft tissue regeneration. The graft copolymer exhibited a lower degree of crystallinity of the PCL and CS chains compared to that found for the two respective homopolymers. This resulted in lower H and E values and a time-dependent behavior of the mechanical properties due to the presence of PCL, but still sufficient integrity under mechanical stress after 3 days of degradation in the cell culture medium α-MEM at 25 °C. The measured E values are close to these reported for soft tissues, rendering the copolymer a suitable candidate for the growth of WJ-MSCs. The in vitro biological evaluation of the materials clearly demonstrated that the CS-g-PCL discs support the growth of WJ-MSCs and lead to a larger than 2-fold increase in proliferation after 3 and 7 days in culture, compared to the PCL samples, and favor tissue formation in culture as indicated by the extracellular collagen production with a simultaneous material degradation. Ongoing investigations on the differentiation of WJ-MSCs to cardiac cells onto 3D CS-g-PCL scaffolds using oxytocin for cardiogenic induction could provide a very promising cell-scaffold construct for myocardium tissue engineering. For this long-term objective, the electrical properties of the graft copolymer should be also studied, as well as the ability of the CS-g-PCL copolymer to induce synchronized beating with the native cardiac tissue.A CS-


In IPLs and PCLS, the pulmonary vessel tone was enhanced with ET-1 or remained unchanged. IPLs were ventilated and PCLS were gassed with argon-mixture or room-air. IPL: Argon reduced the ET-1-induced increase of Pcap, Rpost and oedema formation (p < 0.05). PCLS (rat): Argon relaxed naïve pulmonary arteries (PAs) (p < 0.05). PCLS (rat/human): Argon attenuated the ET-1-induced contraction in PAs (p < 0.05). Inhibition of GABAB-receptors abolished argon-induced relaxation (p < 0.05) in naïve or ET-1-pre-contracted PAs; whereas inhibition of GABAA-receptors only affected ET-1-pre-contracted PAs (p < 0.01). GABAA/B-receptor agonists attenuated ET-1-induced contraction in PAs and baclofen (GABAB-agonist) even in pulmonary veins (p < 0.001). PLCS (rat): Argon did not affect the airways. Finally, argon decreases the pulmonary vessel tone by activation of GABA-receptors. Hence, argon might be applicable in patients with pulmonary hypertension and right ventricular failure.Argon exerts neuroprotection. Thus, it might improve patients’ neurological outcome after cerebral disorders or cardiopulmonary resuscitation. However, limited data are available concerning its effect on pulmonary vessel and airways. We used rat isolated perfused lungs (IPL) and precision-cut lung slices (PCLS) of rats and humans to assess this topic. IPL: Airway and perfusion parameters, oedema formation and the pulmonary capillary pressure (P The protective effects of argon and xenon on cellular integrity have been shown for numerous conditions being at high risk for organ dysfunction or poor cerebral outcome, e.g. cardiac surgery6, cardiac resuscitation10, transplantation11 or neurological disorders18. The mechanisms beyond xenon-induced neuroprotection comprise NMDA-antagonism and activation of two-pore potassium channels (TREK-1) or KATP-channels1. Referring the neuroprotective effects of argon, several mechanisms are discussed; e.g. activation of ERK1/2 and PI3K-AKT20, stimulation of TLR2/422 and up-regulation of the anti-apoptotic gene Bcl-218. Recently, a common mechanism of argon and xenon has been identified. Both noble gases desensitise acid-sensing ion channels which was shown to be neuroprotective in mouse models of ischaemic stroke23. Regarding the anaesthetic effect of argon under hyperbaric conditions, GABAA-receptors appear to be involved24.Noble gases were considered to be inert due to their filled outer electron shell: Meanwhile it is recognised that they exert physiological effects by van der Waals forces25. The clinical application of argon appears to be more conceivable. The fact that it is non-anaesthetic at normobaric pressure might be even advantageous, as patients requiring neuroprotection are rather harmed from additional sedation. Concerning the neuroprotective effects of argon, only experimental data are available thus far. Yet, one study in humans confirmed that short-term exposure to argon does not affect cerebral circulation or metabolism26.The use of xenon is limited due to its rarity of 0.09 ppm in the atmosphere; in contrast argon is abundant at 0.93%28. One trial in newborn pigs assessed the systemic vascular effects of argon and showed that ventilation with argon (80%) does not affect heart rate or mean arterial blood pressure29. Further, Martens et al.28 showed that ventilation with argon (70% or 79%) does not affect total pulmonary vascular resistance (PVR). However segmental PVR, expressed as precapillary (Rpre) and postcapillary resistance (Rpost) gives much more evidence about the pulmonary arterial and venous bed. This topic is even more relevant, as pulmonary arteries (PAs) and veins (PVs) react quite differently32 and PH due to LHD primarily affects PVs34.Usually, neuroprotection is warranted in patients suffering primary neurological disorder  or secondary cerebral ischaemia due to cardiac arrest and cardiac surgery. These patients are often affected by cardiovascular and pulmonary disorders; e.g. left heart disease (LHD), right ventricular (RV) failure, pulmonary hypertension (PH), chronical asthma or chronic obstructive lung disease. Hence, the effects of inhaled argon on airway or pulmonary haemodynamic parameters should be considered. Currently, clinical studies addressing this topic are lacking and experimental trials are rarepre and Rpost in isolated perfused lungs (IPL)35 which allows for the measurement of the capillary pressure (Pcap) and the calculation of Rpre and Rpost. The direct effects of argon and the role of GABA were studied in precision-cut lung slices (PCLS) from rats or humans36. PCLS are viable for 72 hours and enable the real time evaluation of the tone of PAs, PVs and airways37.We studied the effects of argon on RWe evaluated the effects of argon on pulmonary haemodynamic and airway parameters using rat isolated perfused lungs (IPL) and precision-cut lung slices (PCLS) of rats and humans. In both models, healthy lungs with or without ET-1 pre-treatment were studied. In addition, we addressed the role of GABA-receptor activation within argon-induced relaxation in naïve or ET-1 pre-contracted rat PCLS.PA), PVR, Pcap, Rpre, Rpost or oedema formation, indicated by the wet-to-dry ratio  as soon as baseline values were stable for 10 minutes. Again 10 minutes later, argon-ventilation was started. Aside from the contractile effects of ET-1 on the pulmonary vascular bed, ET-1 provokes bronchoconstriction38.To study the effects of argon under conditions of increased PVR and to mimic a feature of PHPA, PVR, Pcap, Rpre and Rpost . Perfusion with ET-1 increased Kfc , PVs and PAs were analysed Fig. . Argon dA-receptor inhibitor gabazine or the GABAB-receptor inhibitor saclofen prior to exposure to argon. Control PCLS only underwent argon-gassing. Regarding IVA, the gabazine/argon group did not differ from the argon group  prevented the effect of argon on ET-1-induced contraction of PAs  also prevented the effect of argon on ET-1-induced contraction  and did not alter the contractile effect of ET-1 in PAs . In PAs, muscimol decreased the contractile effect of ET-1 compared to their exposure only to ET-1  reduced the tone of rat PAs and lowered ET-1-induced contraction in PAs from rats or humans. In IPLs, ventilation with an argon-mixture reduced the ET-1-induced increase of Pcap, Rpost and the W/D-ratio. Regarding argon-induced relaxation, GABA-receptors appear to be involved, as 1) inhibition of GABAB-receptors prevented the relaxant effect of argon in naïve PAs and 2) inhibition of GABAA/B-receptors blocked the attenuating effect of argon on ET-1-induced contraction. Beyond that, GABAA/B seems to interact with ET-1, as stimulation of GABAA/B- or GABAB-receptors reduced ET-1-induced contraction in rat PAs or PVs, respectively. In the IPL, argon exerted some effects on the airway tone which were not confirmed in PCLS and discussed later.In this study, argon relaxed the pulmonary circulation. Gassing with an argon-mixture  ET-1-induced contraction differed among PAs of rats or humans and was about 63% or 25% of IVA, respectively. This fact could explain the delayed effect of argon on ET-1-induced contraction in human PAs. (2) Rat PAs relaxed without pre-contraction suggesting a certain resting tone, as it was shown for PVs from guinea pigs31. (3) Human PAs only relaxed due to argon, if they were pre-contracted, as it was shown for milrinone31 and other relaxant stimuli (our own unpublished data). Most likely, they do not dispose of a resting tone, 4) PAs from both species belong to different parts of the pulmonary arterial bed. PAs from rats derive from a central part of the lung, whereas human PAs derive from a more peripheral part of the lung. Regarding the diverging responses to argon, we can expect species-dependent differences36, but also that various pulmonary vascular segments react differently to similar stimuli42.In rat PAs, argon exerted relaxation and reduced the contractile effect of ET-1. Both effects were evident, if argon-gassing was performed for 2 hours, whereas argon did not alter the tone of PVs, emphasizing the different response of PAs or PVs to various stimuliPA or Rpre, regardless if PVR was increased by ET-1 or not. In the IPL, PPA represents the central part of the pulmonary arterial bed which corresponds to rat PAs studied in PCLS, whereas Rpre rather displays peripheral PAs. So, it was somewhat unexpected that ventilation with argon did not affect PPA in untreated lungs or did not lower the effect of ET-1 on PPA. Regarding these discrepancies, the following ideas should be considered. When PCLS are exposed to argon the argon-mixture reaches the PAs, PVs or AWs directly, thus allowing the study of argon’s effects with confidence. Unlike PCLS, inhaled argon initially must pass the pulmonary vasculature at the capillary and postcapillary level where, argon effectively relaxed the tissue targets. However, it is uncertain, if argon acts in other parts of the pulmonary circulation which are more distant to the alveolo-capillary membrane to the same extent, or whether higher concentrations are required, e.g. realised by an extended application period. Potentially, the pulmonary arterial relaxant effects of argon shown in PCLS are less relevant for clinical practice. Irrespective of these considerations, argon did not deteriorate pulmonary haemodynamics, as it was shown for xenon44. It rather exerted beneficial effects on Pcap, Rpost and oedema formation which are not debatable and of interest in patients with LHD often suffering from postcapillary PH and/or lung oedema45.Despite the intriguing effects of argon on the pulmonary arterial tone of rats or humans, argon did not influence Pcap and prevented the effects of ET-1 on the W/D-ratio and on Kfc. Apart from that, argon reduced the ET-1-induced increase of Rpost resembling the postcapillary vascular bed, and thus the smallest PVs. Our data from PCLS (rats) do not reflect these results, as argon did not relax PVs and did not attenuate the contractile effect of ET-1 in PVs. Nonetheless, they become more distinct, if some other factors are considered. (1) Rpost is determined by the tone of the smallest PVs which are not reflected in PCLS, as PCLS from rats allow the study of central, large PVs, whereas human PCLS enable the study of more peripheral, but not the smallest PVs. (2) PLA reflects the central pulmonary venous system. However, if constant flow is applied during negative pressure ventilation, it is essential to establish a pressure balancing chamber in the perfusion outflow to prevent negative pressure lung oedema. This pressure balancing chamber is connected by tubes to the artificial thorax chamber. Hence, PLA conforms to the pressure in the thorax chamber. Finally, we studied in PCLS central or medium-sized PVs, whereas in the IPL, the smallest parts of the pulmonary venous bed were addressed.In the IPL, argon reduced the ET-1-induced increase of Ppost, but did not influence PVR. At first look this appears to be a discrepancy, but it is explainable. PVR is determined as followed: PVR = (PPA − PLA) × 80/flow. Due to the facts that (1) argon had no effect on PPA, (2) PLA was fixed due to the pressure balancing chamber and (3) the flow was constant, PVR could not change. In contrast, Rpost is calculated as followed: Rpost = (Pcap − PLA)/flow. Based on the facts that argon lowered the ET-1-induced increase of Pcap, whereas PLA and the flow remained constant, Rpost unavoidably decreased. We hypothesise that argon would have decreased the effect of ET-1 on PVR, if we had applied positive pressure ventilation.Here, argon lowered the ET-induced increase of Ret al.27 studied the organoprotective effects of argon in a porcine model of ex vivo lung perfusion. PVR was increased by a warm ischaemic period of 2 hours. Afterwards, argon ventilation was started, but without effects on PVR. In a similar work, Martens et al.28 studied the organoprotective effects of argon within the context of pre-conditioning. Again, argon did not alter PVR. The studies from Martens et al.28 and our study show diverging results and are difficult to compare. (1) Martens et al. increased PVR by a warm ischaemic period, whereas we used ET-1. (2) They used a model without fixed PLA, but in our set-up PLA was fixed. (3) They did not determine segmental PVR  and (4) both studies were done in different species.Other studies addressing the pulmonary vascular effects of argon are scarce. Martens 48 and within the pathogenesis of acute lung injury49. This issue is emphasised by the fact that ET-1-antagonists attenuate the occurrence of acute lung injury52. Here, argon ventilation completely prevented the effect of ET-1 on the W/D-ratio and on Kfc. Thus, argon prevented the formation of lung oedema by a decrease of Pcap which is the main pressure driving fluid from the pulmonary capillaries to the interstitium53 and by decreased vascular permeability. This topic is clinical relevant, as patients suffering to neurological illness often develop neurogenic lung oedema being crucial for their prognosis54. Our results are in conflict to those of Martens et al.28 who did not observe argon-related effects on the W/D-ratio. Possibly, the diverging results rely on the various modes of induction of lung oedema. These conflicting results suggest that argon should be further explored within acute lung injury or lung oedema.In this study, PVR was increased by ET-1 to mimic a characteristic of PH. Notably, increased ET-1-levels play a role in sepsis, sepsis-related organ dysfunction20, TLR2/422 or Bcl-218 is discussed, though all these targets are fairly unspecific. The anaesthetic effect of argon is referred to the activation of GABAA-receptors24. GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian brain55. Beyond that, GABA-receptors are found in the lungs57. There, activation of GABAA-receptors leads to airway smooth muscle relaxation62 and plays a role in the fetal development of the lung64.Argon-induced downstream signalling is unexplored. In view of neuroprotection, a role of ERK1/2, PI3K-AKTA/B-receptors did not affect the tone of naïve PAs and did not alter the contractile effect of ET-1 in PAs. Thus, the basal activation of GABAA/B-receptors appears to be not relevant. However, inhibition of GABAB-receptors (saclofen) reduced the relaxant effect of argon in rat PAs indicating a certain role of GABAB-receptors within argon-induced relaxation. The relevance of GABA for the regulation of the pulmonary vascular tone is supported by Starke et al.65 who proved in PAs from rabbits that their contractile force is reduced, if PAs were treated with GABA and further, that GABAA-inhibition by bicuculline or picrotoxin did not prevent this effect. Hence, a dominant role of GABAB appears to be possible. Here, argon lowered the contractile effect of ET-1 in rat PAs and this effect was prevented, if GABAA/B-receptors were blocked by gabazine or saclofen. These data allow us to conclude that argon relaxes rat PAs via activation of GABAA- and GABAB-receptors. They are in line with data from Kaye et al.66, who found in the feline pulmonary vascular bed that both the GABAA-agonist muscimol and the GABAB-agonist SKF-97541 relaxed the pulmonary vascular bed, if it was pre-contracted with the thromboxane analogue U46619. Regarding the pulmonary vasorelaxant effects of GABA, further studies are lacking. Though, Suzuki et al.67 showed that monocrotaline-induced pulmonary vascular remodelling was attenuated, if rats were pre-treated with GABA leading to decreased levels of norepinephrine. To the best of our knowledge, the presence of GABA-receptors has been proven in the lung57, but not specifically in the pulmonary circulation, even if the activity of GABA-transaminase was verified in PAs or PVs of guinea pigs, with dominance for PAs suggesting the presence of GABA-receptors68. In addition, there is evidence that stimulation of GABA-receptors alters the tone of systemic vessels70.Here, inhibition of GABAfc. Within this context, the role of GABA is supported by several studies indicating its protective effect on the development of lung oedema, e.g. Chintagari et al.71 reported that intratracheal instillation of GABA attenuated the effect of high-tidal volume ventilation on the formation of lung oedema by an increased alveolar fluid clearance. Conversely, this effect was prevented if GABA was instilled together with the GABAA-antagonist bicuculline71. However, there is also evidence that activation of GABAA-receptors aggravate lung oedema64. These contrasting results might be explained by a switch of the Cl− conductance pattern of GABAA-receptors according to the intracellular Cl− concentration63. In addition, Zhang and colleagues72 showed that propofol also acting on GABAA-receptors73 reduces the occurrence of neurogenic pulmonary oedema.Beyond the pulmonary vasorelaxant effects of argon, argon reduced the effects of ET-1 on the formation of lung oedema and on K29 who did not find altered lung mechanics or blood gas analyses due to inhalation of argon. Hence, it must be questioned which phenomena might account for the noticed reduction of TV and C?In the IPL, we found a significant reduction in TV and C with the administration of the argon-mixture. These changes should not be related to the different viscosity of both gases, as (1) we performed a viscosity based correction of our data (explained in the method section) and (2), if we did not correct the data, TV and C would have been rather increased due to the higher viscosity of the argon-mixture. Our results from PCLS do not show any changes of the airway tone due to the exposure to the argon-mixture. They are further in line with othersThe ventilation of the IPL is initiated by a negative pressure in the lung chamber. This amounts to a pressure controlled ventilation mode, such that the TV normally should be the same (based on the lung compliance) independent of the viscosity of the argon-mixture in spite of the change in airway resistance, though the time to filling could increase. However, due to the physiologically realistic breathing frequency of 70 per minute, the increased time to fill might have caused the small difference in TV that was observed.A/B-receptors within the pulmonary vasorelaxant effect of argon, it is somewhat unexpected that argon did not exert bronchorelaxation, as activation of GABAA-receptors was shown to relax airway smooth muscle in several studies62. In view of the pulmonary circulation, it seems that argon-mediated activation of GABAB-receptors is more dominant than stimulation of GABAA-receptors. Anyhow, if argon stimulates GABAA-receptors in PAs, we assume that this should be also the case in AWs. Possibly, the lack of bronchorelaxant effects of argon relies on the intensity of ET-1-induced bronchoconstriction. Obviously, ET-1 contracted PAs and PVs to 60–65% of IVA, whereas the AWs contracted to 10–15% of IAA. Most probably, bronchoconstriction was too strong for a later relaxation.Though, regarding the role of GABAA/B-receptors (muscimol/baclofen) reduced the contractile effect of ET-1. In contrast, in PVs this was only the case, if GABAB-receptors were stimulated. Conversely, muscimol and baclofen did not alter ET-1-induced contraction, if GABA-receptors were blocked with gabazine or saclofen, emphasising the specific activation of GABAA/B. In this view, it must be questioned if ET-1 acts at all on GABAA/B-receptors, or rather if activation of GABAA/B-receptors alters ET-1-induced contraction. From the literature there is some evidence that GABA interacts anyhow with ET-1, e.g. it was reported74 that the application of the GABAA-antagonist bicuculline led to the generation of lung oedema. In that study, ET-1 levels were increased in the bronchoalveolar lavage of bicuculline-treated rats74 and conversely, the occurrence of lung oedema was attenuated by phosphoramidon or by the ETA-antagonist BQ-12374. In addition, GABA reduces the release of norepinephrine67 which highly contributes to the formation of neurogenic lung oedema76.In rat PAs, activation of GABAA/B-receptors plays a pivotal role. Finally, our results support the application of argon for neuroprotection in patients with critical pulmonary haemodynamics based on PH, RV failure or LHD. The relevance of our findings is strengthened by the fact that argon also relaxed human PAs.In conclusion, argon decreased the pulmonary vascular tone of the rat, if PVR was enhanced, but it did not affect the airway tone. In view of the pulmonary vasorelaxant potential of argon, activation of GABAFemale Wistar rats (250 ± 50 g) were purchased from Charles River  and used as lung donors. Rat lungs were randomly assigned to one of the groups.Human PCLS were prepared from patients undergoing lobectomy due to cancer. After pathological inspection, cancer free tissue from a peripheral pulmonary part was used. None of the patients showed any signs of PH . The study was approved by the local ethics committee (EK 61/09) of the Medical Faculty Aachen, Rhenish-Westphalian Technical University Aachen. All patients gave written informed consent.All animal studies and experimental procedures were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz North Rhine-Westphalia  and performed due to the Directive 2010/63/EU of the European Parliament.77. Briefly, female rats were anaesthetised with 95 mgkg−1 pentobarbital  and bled, if reflex checks were unresponsive. The trachea was cannulated and the lungs were ventilated with positive pressure (70 breath/min). In addition, a PEEP of 3 cmH2O and an I:E of 1:1 were applied. As soon, as cannulas were inserted into the pulmonary artery (inflow) and the left atrium (outflow), the lungs were perfused at constant flow (20 ml/min) with 200 ml Krebs-Henseleit buffer, containing 2% bovine serum albumin, 0.1% glucose, 0.3% HEPES and 50 nM salbutamol to prevent bronchoconstriction78. The buffers’ temperature was kept at 37 °C by a water bath and the pH was maintained between 7.35 and 7.45 by gassing with CO2. After pulmonary ventilation and perfusion was established, heart and lungs were removed and set into a negative-pressure chamber which was adjusted for Pmin = −7 cmH2O and Pmax = −2 cmH2O. To prevent lung oedema during constant flow perfusion and negative pressure ventilation, a pressure balancing chamber was set in the perfusion outflow and connected by tubes to the artificial thorax chamber. To prevent atelectasis, every 5 minutes a deep breath was applied. Airway  and pulmonary haemodynamic parameters  were recorded with the Pulmodyn Software 2.0 . If all parameters were stable, the PVR was enhanced with ET-1 79.IPLs were prepared as described beforetp). In the IPL, ΔPtp reflects the difference of the maximal and minimal pressure (P) in the thorax chamber. So, C was calculated: C = TV/(Pmax − Pmin) and R (resistance) was calculated by the inhalation flow (Q) in relation to Pmax and Pmin: R = (Pmax − Pmin)/Q35.The pressure inside the thorax chamber was measured by a pressure transducer . The inhalation flow (Q) was measured by a pneumatograph . Finally, TV was calculated from the integration of Q. The compliance (C) of the lung expresses its elasticity. C is defined by the TV and the change (Δ) of the transpulmonary pressure (Pµ); i.e., if the gas viscosity is increased the pressure drop needed to drive the flow would be increased. The pneumatograph was originally calibrated for air; thus for the argon mixture that has a greater viscosity (2.227 kg/(s-m)10−5 for argon at 32 °C versus 1.84 kg/(s-m)10−5 for air at 32 °C) the measured pressure drop will be greater than that for air at the same flow rate. Therefore, a recalibration of the pneumatograph for the argon-mixture would be necessary; however this is not applicable without interrupting the experiment. Another possibility is to correct the pressure drop data by multiplying by the viscosity ratio of air over argon to obtain the flow rate. Alternatively, TV, R and C can be corrected. We corrected TV, R and C as followed: TVcorr = TVmeas × (µair/µargon-mix); Rcorr = Rmeas × ( µair/µargon-mix) and Ccorr = TVcorr/(Pmax − Pmin)80.To ventilate the lungs with argon, a pressure regulator and a flow meter were used. Argon (flow 0.3 L/min) was applied via a tube which was connected to the pneumatograph. Inhalation flow was measured using a pneumatograph consisting of a single tube with small enough diameter to maintain laminar flow such that the pressure drop across the tube is linearly proportional for the flow rate. The complication in this application occurs because the proportionality is dependent on the gas viscosity  was determined as described in reference35. Measurements were performed at 0 and 120 minutes of the perfusion using the following equation: Kfc = (dweight/dtime)/dPcap. Due to the fact, that weight gain measurements do not allow the simultaneous application of the double occlusion method, Pcap was calculated according to the Gaar equation82: Pcap = PLA + 0.44 (PPA − PLA).To distinguish, if lung oedema derives from increased P36. Rat lungs were filled via the trachea and human lungs were filled via a main or lobar bronchus, respectively, with 1.5% low-melting agarose. Afterwards, they were cooled on ice. Tissue cores (diameter 11 mm) were prepared and cut into about 250 µm thick slices with a Krumdieck tissue slicer . PCLS were incubated over night at 37 °C and repeated medium changes were performed to wash out the agarose.Rats received intraperitoneal anaesthesia with pentobarbital, which was verified by missing reflexes. Thereafter, they were prepared as described beforeA-receptor antagonist gabazine (5 µM)83 or with the GABAB-receptor antagonist saclofen (5 µM)84 86 or the GABAB-receptor agonist baclofen (5 µM)87. In PCLS, all changes of the IVA and IAA were quantified in % and indicated as “Change of IVA [%]” or “Change of IAA [%]”. Thus, an IVA < 100% indicates contraction and an IVA > 100% indicates relaxation. In order to compare the effect of argon in pre-treated vessels, the intraluminal area was defined after pre-treatment again as 100%. In the graphs, all pre-treatments were indicated. The intraluminal area of PAs, PVs and airways was monitored with a digital video camera . The images were analysed with Optimas 6.5 .To study the role of GABA within argon-induced relaxation, PCLS were treated with the GABA)84 Fig. . To stud)84 Fig.  and huma)84 Fig. . To stud)84 Fig.  or with )84 Fig. . To stud)84 Fig. , ET-1 an)84 Fig.  or ET-1 )84 Fig.  prior to2 5%, O2 21%) or air mix , we used the modular incubator chamber  with a filling volume of 6 liters. To avoid evaporation, we added 20 ml of purified water in a petri dish inside the chamber. Next, the incubator chamber with the PCLS inside was purged with the appropriate gas mixture and a gas flow rate of 20 L/min for 5 minutes; the outlet and inlet ports where closed with plastic clamps. Alkalisation of the incubation medium was prevented, as all gas mixtures were enriched with 5% CO2. Afterwards, the whole chamber was transferred in a heat CO2 incubator. In order to investigate the effects of argon on pre-constricted airways or vessels, we pre-treated PCLS with ET-1 and made images at 1 h, 2 h, 3 h, 6 h and even 24 h, in case of human PCLS. To study the role of GABA within the relaxant effect of argon, we pretreated PCLS with the GABA inhibitors gabazine or saclofen and made images according to the pre-treatment with ET-1. Images were recorded by videomicroscopy and the IVA/IAA was calculated with Optimas 6.5.PCLS were transferred into 24 well plates (1 ml medium per well). In order to treat PCLS with argon . The potency of ET-1 differs strongly with age and lot numbers. GABA receptor agonists/antagonists and standard laboratory chemicals were from Sigma-Aldrich . Gas mixtures were delivered from Air Liquide GmbH  or Linde Gas AG .Statistics was conducted using SAS 9.2  and GraphPad Prism 5.01 . All data were analysed by a linear mixed model analysis, except Figs Animal studies were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz North Rhine-Westphalia  and performed due to the Directive 2010/63/EU of the European Parliament.Human PCLS were prepared from patients undergoing lobectomy due to cancer. All patients gave written informed consent and the local ethics committee (EK 61/09) of the Medical Faculty Aachen, Rhenish-Westphalian Technical University Aachen, approved the study.


Guillardia theta at 1.90 and 1.65 Å, respectively. The structures of PEBB exhibit the typical α/β/α-sandwich fold. Interestingly, the open-chain tetrapyrrole substrate DHBV is bound in an unexpected flipped orientation within the canonical FDBR active site. Biochemical analyses of the WT enzyme and active site variants identified two central aspartate residues Asp-99 and Asp-219 as essential for catalytic activity. In addition, the conserved Arg-215 plays a critical role in substrate specificity, binding orientation, and active site integrity. Because these critical residues are conserved within certain FDBRs displaying A-ring reduction activity, we propose that they present a conserved mechanism for this reaction. The flipped substrate-binding mode indicates that two-electron reducing FDBRs utilize the same primary site within the binding pocket and that substrate orientation is the determinant for A- or D-ring regiospecificity.Phycobilins are light-harvesting pigments of cyanobacteria, red algae, and cryptophytes. The biosynthesis of phycoerythrobilin (PEB) is catalyzed by the subsequent action of two ferredoxin-dependent bilin reductases (FDBRs). Although 15,16-dihydrobiliverdin (DHBV):ferredoxin oxidoreductase (PebA) catalyzes the two-electron reduction of biliverdin IXα to 15,16-DHBV, PEB:ferredoxin oxidoreductase (PebB) reduces this intermediate further to PEB. Interestingly, marine viruses encode the FDBR PebS combining both activities within one enzyme. Although PebA and PebS share a canonical fold with similar substrate-binding pockets, the structural determinants for the stereo- and regiospecific modification of their tetrapyrrole substrates are incompletely understood, also because of the lack of a PebB structure. Here, we solved the X-ray crystal structures of both substrate-free and -bound PEBB from the cryptophyte  A similar reduction is catalyzed by the plant FDBR phytochromobilin synthase (HY2), which uses BV as a substrate and catalyzes the two-electron reduction to phytochromobilin 2–omobilin –15. An AV to PEB , 16, 17.Guillardia theta. This organism evolved by secondary endosymbiosis and employs soluble phycobiliproteins in the thylakoid lumen of the chloroplast  from the eukaryotic cryptophyte alga oroplast , 18. Thecterized . The newGtPEBB was determined by single-wavelength anomalous dispersion. We were able to build and refine an initial atomic model for the majority of the protein, but density for the N-terminal extension unique to eukaryotes was absent. Therefore, we designed an expression construct for a truncated version of GtPEBB lacking the first 83 amino acids, which were annotated as chloroplast transit peptide by ChloroP (GtPEBB (mGtPEBB). Crystals of mGtPEBB diffracted slightly better, showing the same space group (P62) and almost identical unit cell parameters. The structure of mGtPEBB was refined at 1.9 Å resolution to an R factor of 21.1% and a free R factor of 23.2% . DHBV is bound in a niche formed by the central β-sheet on the proximal and by helices H6/H7 on the distal side of the substrate (b). When comparing the substrate-free and the substrate-bound GtPEBB structures, only minor conformational changes are observed upon DHBV binding. Both models superimpose nearly perfectly, showing a root-mean-square deviation of 0.31 Å . Slight side-chain movements of several residues lining the substrate-binding pocket are observed . Substrate binding mostly leads to a stronger coordination of the mobile residues between helix H3 and β-strand S7, forming the “lid” of the active site. These residues could not be modeled reliably in the substrate-free structure but showed defined density in the substrate-bound structure.The eukaryotic m-helices a. DHBV iubstrate b. When cb). Although Asp-219 coordinates a central pyrrole water molecule, Asp-99 is in direct contact with the substrate and coordinates the polar pyrrole nitrogen of the D-ring and the carbonyl oxygen of the A-ring. In our crystal structure, Asp-99 had to be modeled in alternate conformations. Interestingly, computational calculations with a modified PROPKA algorithm showed a shift for the paK from below 7 for the two conformations rotated away from the A-ring to above 8 for the conformation in coordinating position toward the carbonyl oxygen . At neutral pH, the latter conformation therefore would have a higher probability to be protonated. This is consistent with the red shift of the absorbance maximum during DHBV binding at neutral pH. In other FDBRs, such a bathochromic shift is attributed to a protonated, positively charged substrate molecule, requiring an adjacent proton donor (GtPEBB–DHBV complex from pH 7.5 to pH 8.5 resulted in a reversible hypsochromic shift consistent with a deprotonation of Asp-99 (data not shown). Because the mGtPEBB–DHBV crystals were grown at pH 8.5 and appeared in similar purple color, the observed alternate conformations most likely reflect this deprotonation of Asp-99. Therefore, we propose the highlighted conformer of Asp-99 as the catalytically active ground state, most likely in a protonated form in the productive enzyme–substrate complex. At the same time, the flexibility of Asp-99 could also be a mechanistic requirement for catalysis as described for PcyA, even though both enzymes differ in their respective catalytic mechanism  with a low root-mean-square deviation of 1.54 Å  . This agrees with the placement of PebBs in a phylogenetic tree close to PebAs , which forms helix 9 and a long loop (23 amino acids) not present in most other FDBR structures . The largest structural differences to PebA are observed for residues 149–151 (GtPEBB numbering), which are part of the lid of the active site and form a short helix in GtPEBB (c). In contrast, no secondary structure elements can be observed in the corresponding stretch of PebA or in PebS . Mostly hydrophobic residues of the central seven-stranded β-sheet build the proximal basis for the binding niche. Asp-99, which is a critical residue in all FDBRs, is located on strand S4. The distal side of the substrate is covered by the two helices H6 and H7 , which are connected by a short loop containing the critical Asp-219, relevant for those FDBRs catalyzing the reduction of DHBV to PEB.The basic geometry of the active site of Synechococcus sp. WH8020 and PebS from the cyanophage PSSM2 were identified as catalytically relevant and show the same orientation toward the substrate center , these result in different substrate conformations and relative positions to the catalytic aspartates. One has to note that especially in the case of PebS, variations of these binding conformations have been observed even within one crystal structure, therefore highlighting an increased flexibility needed for the four electron reduction  was proposed to ensure the stereospecific protonation of C2 at the A-ring, resulting in the R configuration. Asp-231 was believed to be involved in direct coordination of the substrate DHBV to support the protonation by Asp-107 . This results in an inverted positioning of the A- and D-rings. In addition, although the ligand adopts a planar conformation with three of its pyrrole rings, one pyrrole ring is tilted out of the plane. Only the methine bridge with the C15–C16 single bond between rings C and D (specific for DHBV) supports such a rotation, thereby further corroborating our assignment. This orientation is not compatible with the proposed reaction mechanism outlined above, because the central aspartate now is positioned on the wrong side of the A-ring.Contrary to this hypothesis, we identified a flipped binding orientation of DHBV in Fig. S3). This residue is integrated into a hydrogen-bonding network to several surrounding residues conserved among the members of the PebB family. Compensating the narrowing of the niche, an aromatic residue present in most FDBRs (Phe-136 in PebA) is replaced by a small residue in PebBs . This exchange provides enough space to fit in the tilted D-ring of DHBV.We therefore analyzed the binding pocket to identify structural factors governing this new substrate-binding mode and giving insights into the mechanism of A-ring reduction within PebB. Specific to PebB is the strictly conserved Arg-215, which strongly modifies the shape of the binding pocket with its long positive charged side chain when compared with PebA and PebS (asterisk). Replacing Arg-215 with lysine maintained side chain charge and size to some extent but resulted in reduced catalytic activity. Based on the HPLC elution profiles, the amount of produced PEB was only approximately one-quarter of the WT. Interestingly, similar to R215A, this variant showed very ineffective binding as judged by the spectral shift of DHBV absorption. No absorption in the region from 670 to 680 nm was detected for R215K. Because we have to assume that the variant still acts via a radical intermediate, its formation has become rate-limiting. In contrast, in the WT reaction, the rate-limiting step is the conversion of the radical intermediate to the final product PEB, therefore explaining the transient radical accumulation.To further corroborate the role of Arg-215 for the function of PebB, we created several variants at this position. Changing the positively charged side chain to one with hydrophobic character  led to a complete loss of activity . PromineIt is somewhat puzzling that different variants do not show the classical shift in absorption of the substrate upon incubation yet retain significant catalytic activity. Because this spectral shift can be reverted by a pH shift in the WT enzyme (as discussed above), it is not a precise indicator of binding itself but also requires the correct protonation state of the binding niche.One major function of Arg-215 is the variation of the shape of the active site by its long side chain as compared with PebA, enforcing the flipped binding mode of DBHV. A second function is the strong coordination of the catalytically important residue Asp-219 by a salt bridge, present in both the substrate-free and DHBV-bound state. Apparently, this ensures the correct orientation of the critical carboxy group of Asp-219. The corresponding conserved Asp-205 in PebA is not essential for the conversion of BV to DHBV in PebA  (5).aK value and therefore the protonation state of the central Asp-99 and/or Asp-219. Indeed, for some members of the lysozyme family, it has been shown that a conserved arginine residue modulates the catalytic capacity of adjacent carboxylates by introducing a strong shift of their paK values while also stabilizing the active site structure  indeed is strictly conserved within the complete PebB and HY2 families  and PebS (methionine). We therefore analyzed the variants R215S and R215M of mGtPEBB with our standard assay, supplying either DHBV or BV to investigate how these strictly conserved residues are affecting substrate specificity  and an increase in peak intensity indicated binding of this unnatural substrate. This is in accordance with results reported for the cyanobacterial PebB  by Arg-215. For the second protonation step, it remains unclear which molecular determinants govern the protonation of the vinyl group at the C1 position. One possible scenario is the reprotonation of the A-ring nitrogen, uptake of another electron, and a final tautomerization to yield the product PEB. Again, the position of the axial water makes it a likely determinant for the stereospecificity of this step.For PebB, the following reaction mechanism would be consistent with our structural and biochemical data of variants . After bGtPEBB mechanistic model outlined above. This again supports a binding mode of BV with the A-ring in the primary site in the case of HY2. Even more interesting, HY2 from ferns or mosses have retained the canonical aspartate pair, supporting their placement close to the base of the evolutionary tree of FDBRInterestingly, this proposed mechanism would be in agreement with the biochemical data available for HY2, which catalyzes the identical reduction on the A-ring (but on BV instead of DHBV) . In addiGtPEBB raises the question of whether BV conversion to PEB functions in the same mechanistic manner in the PebA/PebB system and in PebS. Available crystal structures show BV bound in the same orientation in both PebA and PebS, leading to the conclusion that they share the reaction mechanism for the first two-electron reduction on the D-ring. This is supported by the biochemical data.The flipped binding mode of DHBV in If A-ring reduction in PebS works in the same mechanistic manner as in PebB, DHBV would have to be released after the first reduction and bound again in the flipped orientation. One can speculate whether this would be realized by a metabolic channeling between two PebS molecules, as described for PebA and PebB , 12.Until now, there is no evidence that the intermediate DHBV is released from PebS after the first reduction. Because the binding niche of PebS provides somewhat more space than in PebA, this would allow a rearrangement of the intermediate DHBV but would still retain the placement of the A-ring in the second site  . ClearlyAll chemicals were ACS grade or better unless specified otherwise. The Assay components including BV were purchased from Sigma–Aldrich.GtPEBB contains a predicted signal peptide of 18 residues and a predicted targeting sequence including residues 19–83. The protein without the signal peptide is referred to as GtPEBB (for consistency with the published construct)  according to the manufacturer's instructions. The vector pGEX-4T-1  with an additional tobacco etch virus (TEV) protease recognition site was linearized by PCR amplification. The plasmid pGtPEBB   before ligation by the Gibson Assembly Master Mix and subsequent transformation into NEB® 5-alpha competent E. coli cells. The resulting constructs contain GtPEBB/mGtPEBB as translational fusion to GST with a TEV site following the thrombin site.The genome-derived sequence of GtPEBB were constructed by PCR site-directed mutagenesis using the primers listed in Table S3. Because the reverse primer is complement, only the forward primer is shown. Introduced bp changes are underlined. Variants R215A, R215L, and R215K were constructed using the QuikChange® Lightning kit (Agilent) according to the manufacturer's protocol. PCRs for R215M, R215S, D99N, and D219N were set up using the PfuUltra II Fusion HS polymerase (Agilent). After DpnI digestion, aliquots of the reaction were transformed into NEB® 5-alpha competent E. coli cells. All resulting plasmids were checked by sequencing.All variants of mGtPEBB and mGtPEBB and variants, 2× YT medium containing 100 μg/ml ampicillin was inoculated 1:100 with an overnight preculture carrying the respective expression plasmid. 1-liter cultures were grown in a 5-liter flask at 37 °C and 100 rpm to an A600 of 0.7 followed by a temperature shift to 30 °C before induction with 300 μm isopropyl-β-d-thiogalactopyranosid. After 4 h expression, the cells were harvested by centrifugation  and stored at −20 °C for further use. Thawed pellets were homogenized in 10 ml of lysis buffer  per gram of cells, and a spatula tip of DNase I (Sigma–Aldrich) was added prior to cell disruption with a microfluidizer M-100L (Microfluidics) at 13,000 p.s.i. After ultracentrifugation , the supernatant was filtered through a 0.2-μm filter and loaded onto a PureCube GSH-agarose (Cube Biotech) column. After washing, bound protein was eluted with elution buffer . Cleavage of the fusion protein was done with a GST-tagged TEV protease (1 mg of GST-TEV per 50 mg of fusion protein) overnight during dialysis against a 200-fold volume of lysis buffer in a 6,000–8,000-Da molecular mass cutoff tubing (Spectrum Labs). The protease digest produced the protein of interest with three additional amino acids at the N terminus  or two additional N-terminal amino acids  for mGtPEBB. To remove residual GST and GST-TEV, the dialysate was loaded again on the GST affinity column. The flow through was collected and run on a HiLoadTM 16/600 SuperdexTM 75-pg  size exclusion column equilibrated with low-salt buffer  for crystallization experiments or 25 mm TES/KOH, pH 7.5, 100 mm KCl for activity assays. All chromatography steps were carried out on an ÄKTA purifier 100 system . The protein was concentrated using Vivaspin® centrifugal concentrators with a 10,000-Da molecular mass cutoff (Sartorius AG).For recombinant production of Synechococcus sp. WH8020 was expressed and purified as described before with slight modifications  instead of a French press and ultracentrifugation were done as described above for PebB. For GST affinity chromatography PureCube GSH-agarose resin (Cube Biotech) was used. Final buffer exchange was done by gel filtration on a HiLoadTM 16/600 SuperdexTM 75-pg column  instead of dialysis. Prior to snap freezing PebA in liquid N2 for storage at −80 °C, the solution was supplemented with 10% glycerol. Ferredoxin from Synechococcus sp. PCC7002 was expressed and purified as described in Ref. PebA from ications . The celGtPEBB the standard protocol described for GtPEBB was modified according to Refs. l(+)-selenomethionine: 50 mg/liter) was added to the culture. All buffers used during purification contained 10 mm DTT.For expression of selenomethionine containing GtPEBB were screened by the sitting-drop vapor-diffusion method applying 100/100- and 200/100-nl mixtures of protein solution (7–10 mg/ml)/reservoir solution incubated at 18 and 4 °C in the dark. Initial crystals grew in 0.1 m Tris pH 8.5, 35% PEG 3350, and 0.2 m ammonium sulfate  at 4 °C. Final crystals of selenomethionine substituted GtPEBB were obtained by optimization of the initial condition of the native protein by the hanging-drop vapor-diffusion method in a 2/1-μl drop of 7 mg/ml protein and 0.1 m Tris, pH 8.5, 34.5% PEG 3350, 0.21 m ammonium sulfate reservoir solution after 4 days at 4 °C.Crystallization conditions for GtPEBB was optimized, and final crystals grew in a 1/1-μl hanging drop over a reservoir solution containing 0.1 m Tris, pH 8.5, 34.5% PEG 3350, 0.15 m ammonium sulfate. For crystallization of the mGtPEBB–DHBV complex, protein solution was incubated with a 2-fold molar excess of DHBV dissolved in DMSO for 1 h on ice in the dark. The complex and free DHBV were separated by applying to a NAPTM-5 column . To achieve stabilization of DHBV during crystal growth, sitting-drop grid screen refinement plates were resealed and stored in an anaerobic chamber CLc (Coy Laboratory Products) after setting them up under standard conditions. Final purple colored crystals grew in a 200/100-nl mixture of protein (8 mg/ml)/reservoir  at 6 °C in the dark. Prior to freezing in liquid N2, all crystals were briefly soaked in mother liquor supplemented with 16% PEG 400 for cryoprotection.Crystallization of mGtPEBB was radiation damage corrected by zero-dose extrapolation in XSCALE. Statistics of all data sets are listed in Oscillation data were collected at 100 K at the Swiss Light Source  at Beamline PXII–X10SA and at the European Synchrotron Radiation Facility  at Beamline ID30B using PILATUS 6M detectors (DECTRIS). All data were processed and scaled using XDS and XSCALE . The datGtPEBB in 7 fragments with Phenix.autobuild . For high redundancy and low dose, the data were collected covering 720° rotation range. Ten selenium sites were refined with Phenix.autosol  of whichutobuild . This inutobuild  and Phenutobuild , but cleGtPEBB was then used for refinement against the native 1.9 Å data set of mGtPEBB, which was obtained by scaling two data sets from individual crystals. The final model of the substrate-free protein was deposited in the PDB with the accession code 6QWQ. Because of weak or missing density, the first three residues and residues 45–48 and 147–151 have been omitted.The initial model of selenomethionine labeled GtPEBB–DHBV complex crystallized in a different space group, we used the substrate-free structure as a search model for molecular replacement with Phenix.phaser , residues 44–49 and the last residue were not modeled because of missing density. Because the overall quality of chain B (including a fully occupied DHBV) was better than that of chain A , we used chain B for all figures.Because the mx.phaser . Geometrx.phaser . The modx.phaser  and Phenx.phaser  and was m TES/KOH, pH 7.5, 100 mm KCl, 10 μm FDBR, 10 μm substrate (BV/DHBV), 10 μm BSA, 100 mm glucose, 50 units/ml glucose oxidase, 50 units/ml catalase, 1 μm ferredoxin from Synechococcus sp. PCC7002, and 0.01 μm ferredoxin NADP+ reductase from Synechococcus sp. PCC7002. Catalysis was started by the addition of 100 μl of a NADPH regenerating system , ending up with a final assay volume of 2 ml (Anerobic bilin reductase assays were performed as described before with slight modifications . Assay c of 2 ml .Utilizing a 8453 series diode array spectrophotometer (Agilent), spectra were taken every 30 s for 10 min. The reaction was stopped by the addition of a 10-fold volume of 0.1% TFA. Bilins were extracted from the reaction mixture using C18 Sep-Pak light cartridges (Waters), which were preconditioned as described before . After em formic acid as the mobile phase  with a Luna 5μ C18 reversed-phase column (Phenomenex) and 50% acetone and 50% 20 mle phase .Synechococcus sp. WH8020 was used for enzymatic production of DHBV for GtPEBB activity tests and co-crystallization experiments. To obtain higher yields, the activity assay was modified by feeding 2–3× new BV after no residual BV was detectable by UV-visible spectroscopy (m Pi, pH 6.5, as the mobile phase. Fractions with pure DHBV were again loaded on a Sep-Pak light cartridge before lyophilization. The lyophilizate was dissolved in DMSO for further experiments.PebA from troscopy . The eluaK values of titratable groups with the residue paK application included in the Molecular Operating Environment platform with default settings (aK is based on PROPKA, but modifications allow that the calculation is not restricted to titratable groups of standard amino acid side chains (Protonation states were assigned by predicting the psettings . Residuee chains , 35.J. A. S., N. F.-D. and E. H. conceived and designed the study. J. A. S. performed biochemical experiments, crystallized the proteins, and solved the structures. J. A. S. and E. H. analyzed the structural data. J. A. S., N. F.-D., and E. H. wrote the manuscript and agreed on the final version of the manuscript.


The Fremantle Back Awareness Questionnaire (FreBAQ) claims to assess disrupted self-perception of the back. The aim of this study was to develop a German version of the FreBAQ (FreBAQ-G) and assess its test-retest reliability, its known-groups validity and its convergent validity with another purported measure of back perception.The FreBaQ-G was translated following international guidelines for the transcultural adaptation of questionnaires. Thirty-five patients with non-specific CLBP and 48 healthy participants were recruited. Assessor one administered the FreBAQ-G to each patient with CLBP on two separate days to quantify intra-observer reliability. Assessor two administered the FreBaQ-G to each patient on day 1. The scores were compared to those obtained by assessor one on day 1 to assess inter-observer reliability. Known-groups validity was quantified by comparing the FreBAQ-G score between patients and healthy controls. To assess convergent validity, patient’s FreBAQ-G scores were correlated to their two-point discrimination (TPD) scores.3.1 = 0.88 (95%CI: 0.77 to 0.94) and 0.89 (95%CI: 0.79 to 0.94), respectively. Intra- and inter-observer limits of agreement (LoA) were 6.2 (95%CI: 5.0–8.1) and 6.0 (4.8–7.8), respectively. The adjusted mean difference between patients and controls was 5.4 . Patient’s FreBAQ-G scores were not associated with TPD thresholds .Intra- and Inter-observer reliability were both moderate with ICCThe FreBAQ-G demonstrated a degree of reliability and known-groups validity. Interpretation of patient level data should be performed with caution because the LoA were substantial. It did not demonstrate convergent validity against TPD. Floor effects of some items of the FreBAQ-G may have influenced the validity and reliability results. The clinimetric properties of the FreBAQ-G require further investigation as a simple measure of disrupted self-perception of the back before firm recommendations on its use can be made. Low back pain is a major cause of disability worldwide  and is aThe cortical body representation is distorted in people with persistent pain which may play an important role in the development and/or maintenance of pain . Early wThese changes can present clinically as alterations in a person’s body perception –13, decrThe Freemantle Back Awareness Questionnaire (FreBAQ) is a simple tool that claims to assess back-specific altered body perception . It comprho = 0.40, p = 0.004) .2 = 0.22). The adjusted mean scores for the patient group was 9.1 (95%CI: 7.77 to 10.87) and 3.7 (95%CI: 2.31 to 5.18) for the control group with an adjusted mean difference of 5.4 .There was a statistically significant effect between groups regarding the FreBAQ-G scores after adjusting for age, gender and BMI (F (1.78) = 20.39, p<0.001, adjusted Rrho = -0.05, p = 0.79) in the patient group  to those reported for the Japanese [3,1 of 0.81 (95% CI: 0.67–0.89)), Dutch [2.1 = 0.69 (95%CI: (0.51–0.82)) and original English version [2.1 = 0.65 (95% CI: 0.307–0.848)). A systematic bias of one unit between time 1 and 2 for observer 1 indicated some small learning effects, thus a familiarization session may be warranted when using this questionnaire.The FreBAQ-G showed moderate intra and inter-observer reliability with ICCJapanese  , Dutch  (ICC2.1  version  (ICC2.1 The SEM (intra- and inter-observer) in our study was ~2units, below the SEM of 3.5 reported by Janssens et al . HoweverTo understand if the FreBAQ-G has sufficient reliability for research purposes it can be useful to use the estimated variability of the measure and its minimally clinically important difference (MCID) to calculate sample sizes for different study designs. There is no existing empirically derived MCID for the FreBAQ. Using 0.5 of a standard deviation as a clinically worthwhile change one could estimate an MCID of approximately 3.0 units for power calculation purposes. Assuming the SD of change is 3.15 see  it can bThe convergent validity of the FreBAQ-G was assessed by correlating it with TPD. There was no correlation between the FreBAQ-G and the TPD, in contrast to our initial hypothesis. These results were in keeping with Wand et al  who founBody perception as measured by the FreBAQ-G was correlated with a number of the clinical outcomes assessed. This implies that the FreBAQ-G may have clinical utility and body perception may be a clinically relevant construct in this patient population. In our study sample, disturbed body perception was associated with pain interference scores (BPI-I), but not with symptom duration. In addition, FreBAQ-G scores showed moderate correlations to back related disability (RMDQ) and anxiety and depression scores (HADS). It may be possible that an altered self-perception of the back, in particular motor neglect aspects, might contribute to motor control impairments, resulting in higher back related disability scores , 55. In Our findings are partly in line with both English study samples, where statistically significant correlations to pain severity were found  and  using the original FreBAQ . This diRegarding the translation process, an initial pre-testing phase in a smaller sample of patients with CLBP could have been utilized to reveal and resolve any difficulties regarding comprehensibility and completeness of contents before commencing the study. However, patients were satisfied with all usability aspects. In addition, we did not measure the exact amount of time it took patients to complete the questionnaire though participants reported that the time to complete was appropriate in their opinion. The current version of the FreBAQ-G demonstrated evidence of floor effects on an item level. This might have adversely affected reliability and validity scores. However, regarding sum scores, the percentage of respondents scoring 0 were below the pre-defined cut-off value of 15%. Hence, our main criterion demonstrated that floor effects did not appear to be an issue in our sample.Although all the patient participants in our study were patients accessing a health care setting for treatment of their CLBP they were on the low end of the spectrum for the range of clinical measures that were used, especially regarding anxiety and depression scores. Thus, our findings may not be generalisable to the wider CLBP population, especially those scoring higher on the clinical spectrum.Our final sample size of 35 patients was lower than current recommendations of 40 participants or more for reliability studies . InitialTo assess the convergent validity of the FreBAQ-G, its scores were correlated to TPD performance which claims to measure the same/or similar construct. The choice of comparator measure to assess convergent validity was difficult as there is no gold standard measure for the construct of self-perception of the back. A recent systematic review published by our group  found thThe translation and assessment of the German FreBAQ is an important step in the use of this questionnaire in people with CLBP, as is makes it available to a German speaking population of 118 million people . The FreThe FreBAQ-G is not proposed as an alternative outcome measure to established clinical measures such as pain and function. However, if a researcher/clinician wishes to assess the specific construct of self-perception of the back very few instruments are available and the clinimetric properties of those measures are limited . If selfWe created a German translation of the FreBAQ. The FreBAQ-G demonstrated a degree of reliability and known-groups validity, while it did not demonstrate convergent validity against a measure, which purports to assess the same construct. These findings are broadly in keeping with other language versions of the questionnaire. The clinimetric properties of the FreBAQ-G require further investigation as a simple measure of self-perception of the back.Given the degree of measurement error the FreBAQ-G could potentially be employed for research purposes to assess back self-perception but it may be too variable to monitor change in individual patients. To minimize learning effects, a familiarisation trial should be considered. The validity of the FreBAQ-G requires further exploration.S1 File(PDF)Click here for additional data file.S1 TableFreBAQ = Fremantle Back Awareness Questionnaire; TPD = Two-Point Discrimination; HC = Healthy controls.(DOCX)Click here for additional data file.


Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases, which is affected by several factors. Anti-histamines, steroids, and immunosuppressive agents have been used for the treatment of AD. However, many studies have reported that long-term use and abuse of these drugs causes many side effects. This study was performed to evaluate the ameliorative effect of green tea extract on AD-like lesions in NC/Nga mice. Green tea extract from tannase digest (GTT), beta-hexosaminidase, and histamine were measured in IgE-antigen complex-stimulated RBL-2H3 cells. Dorsal skin application of house dust mite-ointment induced AD-like symptoms in NC/Nga mice. Dermatitis scores, skin moisture, transepidermal waterloss (TEWL), thickness of skin and ear, T-cell proliferation, levels of immunoglobulins and cytokines, and infiltration of mast cell were measured to assess the degree of AD induction. Skin moisture and TEWL were measured using probes, and ELISA was performed to measure the immunoglobulin and cytokine levels in blood. GTT was selected based on its ability to inhibit the release of beta-hexosaminidase and histamine in IgE-antigen complex-stimulated RBL-2H3 cells. Oral administration of GTT significantly suppressed the skin inflammation and symptoms of AD-like skin lesions in NC/Nga mice. GTT may have a potential therapeutic effect in the treatment of AD. The skin plays an important role in the body defense against harmful stimuli. Atopic dermatitis (AD) is a chronic inflammatory skin disease that causes severe itching and dry skin after contact with aeroallergens such as house dust mites, pollen, and animals . Atopic The treatment of AD involves the use of steroids, anti-histamines, and immunosuppressive agents . SpecifiArtemisia capillaris exerted inhibitory effects on AD-like skin lesions via the downregulation of serum histamine content and IgE expression  method was used for the cytotoxicity assay. As shown in Fig. The inhibitory effect of green tea extracts on IgE-mediated degranulation in RBL-2H3 cells was measured using β-hexosaminidase and histamine secretions as degranulation biomarkers. The release of β-hexosaminidase and histamine in IgE-antigen complex-stimulated RBL-2H3 cells was significantly increased compared with control cells. Pre-treatment with 1 µM of dexamethasone, the positive control, significantly suppressed the degranulation in IgE-antigen complex-stimulated RBL-2H3 cells. The GTT effectively reduced the release of β-hexosaminidase and histamine at all concentrations in RBL-2H3 cells stimulated with IgE-antigen complex  antigen for 28 days. After 14 days, dermatitis scores increased due to the induced AD, and oral administration of GTT resulted in a dose-dependent reduction in the dermatitis scores. The positive control (5 mg/kg of prednisolone) as well as 400 mg/kg of GTT significantly decreased the dermatitis scores during the experimental period.When AD gets worse, the skin moisture was decreased. We analyzed the skin moisture content and transepidermal waterloss (TEWL). On day 14, the moisture capacity of the dorsal skin of mice was decreased by the induced AD and prednisolone, and high concentrations of GTT resulted in a significant recovery of the skin moisture. After 21 days, the hair of the dorsal skin interrupted the measurement of skin moisture, so the skin moisture measurements could not be carried out. However, TEWL was measured throughout these experiments, which increased with HDM and decreased by oral administration of prednisolone and GTT. GTT significantly decreased the TEWL in a dose-dependent manner.As shown in Fig. On day 28, we killed the AD-induced mice and the weight of their spleen was measured. Spleen of the AD-induced mice was bigger compared with the control. However, prednisolone and 400 mg/kg GTT treatments resulted in recovered normal weight of the spleen. The inhibitory effect on the proliferation of splenocytes by GTT was determined by MTT assay. LPS stimulates B-cell proliferation and ConA stimulates T-cell proliferation. Both prednisolone and GTT did not affect the LPS-induced splenocyte proliferation, but ConA-induced splenocyte was significantly enhanced by the induced AD and significantly decreased by the administration in a dose-dependent manner. These results suggested that the AD might be related to stimulation of T cells and GTT could alleviate the AD by inhibiting the stimulation of T cells Fig. .To determine whether orally administered GTT affects the production of immunoglobulins, cytokines, and histamine, blood was collected from the mice. Application of HDM antigen resulted in a high concentration of IgE in NC/Nga mice. Compared with the control group, IgE concentration was significantly downregulated in prednisolone and 400 mg/kg GTT-fed groups. Otherwise, IgG2a was decreased by the induced AD and significantly increased by the administration of prednisolone and 400 mg/kg GTT. The amounts of Th1 cytokine IFN-γ and Th2 cytokine IL-4 in the blood did not differ significantly by the induction of AD. The histamine level of blood was significantly increased by the induced AD and decreased by the administration of 400 mg/kg GTT, but there was no inhibitory effect of the positive control, prednisolone Fig. .Figure Many researchers are constantly studying extraction techniques to improve the functionality and taste of tea , 13, 24.In general, AD is an immunological disease resulting from an imbalance favoring type-2 helper T (Th2) cell. Th2 cells, mast cells, or other immune components cells produce interleukin-4 (IL-4), which is known to induce B-cell class switching to IgE , 26, 29.Likewise, mast cells are the primary effector cells involved in the allergic inflammatory reaction. The degranulation process starts by the interaction of antigens with mast cells . In addiThe NC/Nga mouse has a mutation on chromosome 9, which is related to the increased production of IgE and a Th2-dominant inflammation . HoweverAtopic dermatitis is an inappropriate immune response, involving an excessive Th2 response . This stIn conclusion, orally administered green tea extract from tannase digest (GTT) suppressed the inflammation of AD-like skin lesions, and decreased the immune cell infiltration into the skin. These results suggest that GTT may have a therapeutic potential in the treatment of AD.